A membrane-associated thyroid hormone binding protein (p55) has previously been purified to homogeneity from cultured A431 human epidermal carcinoma cells. Partial sequences from the N-terminal and a cyanogen bromide fragment of p55 were determined. One polyclonal and four monoclonal antibodies against p55 were used to screen the Lambda-gt11 cDNA libraries prepared from human A431 and KB cells; 22 and 13 positive plaques were obtained, respectively. The size of the insert ranges from 0.5-1.8 Kb. Using 1.8 Kb insert as a probe to screen a human cDNA library from fibroblast cells, a clone with a 2.2 Kb insert was obtained. This size is similar to that of mRNA of p55 determined by Northern blot analysis. The 5' sequence of 2.2 Kb fragment was determined which contains the initiation codon and the N-terminal sequence of p55. The apparent full-length cDNA clone for the thyroid hormone binding protein is being characterized. Analysis of the cellular extracts of A431 cells indicated the presence of another binding protein for 3, 3', 5-triiodo-L-thyronine (T3). In contrast to p55, this binding component has an apparent MW of 58K and a PI of 7.1. By a combination of Sephadex G-200, OAE, SP-Sephadex and hydroxyapatite column chromatography, p58 was purified to near homogeneity. Antibodies to p58 are being prepared and its cellular functions will be evaluated.